Page 1 of 31

Virome of >12 thousand Culex mosquitoes from throughout California

Mohammadreza Sadeghi1,2,3; Eda Altan1,2; Xutao Deng12; Christopher M. Barker4, Ying

Fang4, Lark L Coffey4; Eric Delwart1,2

1Blood Systems Research Institute, San Francisco, CA, USA.

2Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA.

3Department of Virology, University of Turku, Turku, Finland.

4Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of

California, Davis, CA, USA.

§Reprints or correspondence: Eric Delwart 270 Masonic Ave. San Francisco, CA 94118 Phone: (415) 923-5763 Fax: (415) 276-2311 Email: [email protected] Page 2 of 31

Abstract

Metagenomic analysis of mosquitoes allows the genetic characterization of all

associated , including arboviruses and insect-specific viruses, plus those in their

diet or infecting their parasites. We describe here the virome in mosquitoes, primarily

Culex pipiens complex, Cx. tarsalis and Cx. erythrothorax, collected in 2016 from 23

counties in California, USA. The nearly complete of 54 different species,

including 28 novel species and some from potentially novel RNA and DNA viral families

and genera, were assembled and phylogenetically analyzed, significantly expanding the

known Culex-associated virome. The majority of detected viral sequences originated

from single-stranded RNA viral families with members known to infect insects, plants, or

unknown hosts. These reference viral genomes will facilitate the identification of related

viruses in other insect species and to monitor changes in the virome of Culex mosquito

populations to define factors influencing their transmission and possible impact on their

insect hosts. Page 3 of 31

Introduction

Mosquitoes transmit numerous arboviruses, many of which result in significant

morbidity and/or mortality in humans and animals (Ansari and Shope, 1994; Driggers et

al., 2016; Gan and Leo, 2014; Reimann et al., 2008; Weaver and Vasilakis, 2009). The

mosquito genus Culex is comprised of ~768 taxa, including some of the most ubiquitous and important vectors of human pathogens which, in the present context of environmental changes affecting their geographic range, pose particular concern

(Harbach, 2011; Jansen et al., 2009; Lequime and Lambrechts, 2014; Li et al., 2010;

Parra-Henao and Suarez, 2012; Wang et al., 2011). The human pathogens vectored by

Culex mosquitoes include West Nile Virus (WNV), Japanese encephalitis virus

(Paraskevis et al.), western equine encephalomyelitis virus (WEEV), and St. Louis encephalitis virus (SLEV). Filarial worms and avian malaria parasites are also transmitted by Culex mosquitoes.

Recent studies have shown the Culex virome to be quite diverse. An early viral metagenomics study revealed DNA viruses as well as mammalian papillomavirus and anellovirus and plant viruses (presumably from the ingested vertebrate blood and plant diet) present in 480 female mosquitoes from multiple mosquito species, including Cx.

erythrothorax from 3 sites in southern California (Ng et al., 2011). Auguste et al.

screened 300 Culex mosquito pools collected in Trinidad for induction of viral cytopathic

effects (CPE) in cultures of C6/36 (Aedes albopictus) mosquito cells. CPE was initiated

by one pool of Cx. declarator mosquitoes collected in Trinidad from which the complete

genomes of two novel insect-specific viruses belonging to the family Bunyaviridae were

sequenced (Auguste et al., 2014). Another study used RNA sequencing to characterize Page 4 of 31 the viral communities associated with 3 mosquito species in northern California: Cx.

pipiens, Culiseta incidens, and Aedes sierrensis. Viral sequences from the families

Bunyaviridae, , and were detected (Chandler et al., 2015;

Coffey et al., 2014; Cook et al., 2013; Cook et al., 2009). A new was identified

from Mansonia africana nigerrima mosquito in Uganda after C6/36 Aedes cell line

amplification (Cook et al., 2009). Using metagenomics, long RNA viral fragments from

diverse families and 2 nearly complete RNA virus genomes were sequenced from

unspecified mosquitoes from Southern France (Cook et al., 2013). Four novel RNA

viruses and other previously sequenced viral genomes were amplified from Australian

mosquito pools by cytopathic effect detection in vertebrate cells (Coffey et al., 2014).

Novel RNA viruses amplified in C6/36 Aedes cells inoculated with different mosquito

pools from Southeast Asia and the Americas were also sequenced describing the

of three new reoviruses and previously described insect viral genomes

(Sadeghi et al., 2017).

Insect-specific viruses (ISVs) and their potential role in disrupting pathogen

transmission has been investigated during the last decade (Bolling et al., 2015;

Calzolari et al., 2016; Nunes et al., 2017; Roundy et al., 2017; Vasilakis et al., 2013;

Vasilakis and Tesh, 2015). Persistent infection of mosquitoes with some ISVs appears

to interfere with the replication and transmission of medically significant viruses, such as

West Nile Virus (WNV) (Bolling et al., 2012; Goenaga et al., 2015; Hall-Mendelin et al.,

2016; Hobson-Peters et al., 2013). The majority of ISVs have been described in

mosquitoes, although they occur in other arthropod orders, including Hemiptera and

Parasitiformes (Li et al., 2015a; Tokarz et al., 2014). ISVs belong to taxonomically Page 5 of 31 diverse virus families including Bunyaviridae, , , Rhabdoviridae,

Togaviridae, , , and (Attoui et al., 2005; Auguste

et al., 2014; Bolling et al., 2011; Calzolari et al., 2016; Ergunay et al., 2017; Fauver et

al., 2016; Huang et al., 2013; Huhtamo et al., 2014; Huhtamo et al., 2009; Kuwata et al.,

2011; Kuwata et al., 2013; Nasar et al., 2012; Schuster et al., 2014).

It is also possible that medically important arboviruses evolved from ISVs that

acquired the ability to infect vertebrates (Vasilakis and Tesh, 2015). Some viruses may

also adapt to animals or plants hosts and lose the need for an insect vector (Li et al.,

2015a). Many insect-associated RNA viruses in the families Bunyaviridae, Flaviviridae

and Rhabdoviridae belong to highly diverse lineages indicating they likely evolved and diversified with their insect hosts over extended time periods (Chandler et al., 2015;

Cook et al., 2013; Marklewitz et al., 2015; Walker et al., 2015). That many of these insect viruses appear to be vertically transmitted without obvious negative fitness consequences may also be considered evidence of a long-term relationship with their insect hosts (Lequime et al., 2016; Marklewitz et al., 2015; Walker et al., 2015). Some viral genomes have also become endogenized in the genomes of their arthropod hosts

(Ballinger et al., 2013; Crochu et al., 2004; Fort et al., 2012). ISVs may also act as natural regulators of insect populations and may provide new avenues for developing vector control strategies. Culex mosquitoes including Cx. quinquefasciatus infected with

the Wolbachia bacteria have been found to influence WNV transmission by lowering

virus titers (Glaser and Meola, 2010).

We sought here to generate a more complete characterization of the Culex

virome using deep sequencing of viral particle-enriched nucleic acids and by sampling a Page 6 of 31 greater number of mosquitoes from a large geographic region. We identified and assembled nearly complete genomes of previously known as well as multiple previously uncharacterized RNA and DNA viruses, compared their genome organizations, and performed phylogenetic analyses. The geographic distribution of these viral genomes throughout California was described. We estimate that the Culex mosquitoes in

California harbor viruses belonging to at least 21 RNA and DNA viral families, as well as several newly described or still unclassified families of DNA and RNA viruses. Page 7 of 31

Materials and Methods

Mosquito Collection and Screening for Arboviruses. The mosquitoes analyzed here

originated from mosquito control districts throughout California. Mosquitoes were initially

collected in carbon dioxide-baited light or gravid traps, morphologically identified to

species by mosquito control district staff, and female mosquitoes were pooled in groups

of 1 to 50 individuals (except one pool of 167 Culicoides sonorensis). Pools were

frozen at -80°C, then shipped on dry ice to the Davis Arbovirus Research and Training

laboratory at the University of California, Davis (UC Davis). There, mosquitoes were

thawed at room temperature, and two glass beads were added to each tube, along with

diluent containing 10% fetal bovine serum and antibiotics (penicillin, streptomycin, and

mycostatin). Each pool was then mechanically homogenized for three min using a dual

mixer mill model 8000D (Spex SamplePrep, Metuchen, NJ) to release virus particles

from mosquito carcasses. The resulting mosquito pool homogenate was centrifuged,

and viral nucleic acids were then extracted from an aliquot of each mosquito pool’s

supernatant using a MagMAX Express-96 Deep Well Magnetic Particle Processor and

then tested by RT-qPCR to detect viral for the three Culex-borne human-

pathogenic viruses endemic to California, WNV, WEEV, and SLEV using a triplex assay

(Brault et al., 2015). Pools that tested negative by RT-qPCR for WNV, WEEV and SLEV

were selected for this study. These mosquitoes originated from 124 unique geographic

locations and represented three different time periods corresponding to early, middle,

and late summer of 2016 (Supplemental Table 1A). 410 pools were assembled into 51

larger pools (Supplemental Table 1B). The total number of mosquitoes from different Page 8 of 31 species analyzed are listed (Supplemental Table 1B), along with the mosquito species in each pool (Supplemental Table 1C).

Deep Sequencing. Mosquitoes from each pool were further homogenized after assembly into larger pools in 1 ml of mosquito diluent (PBS) with disruption beads

(0.16g of 2.3mm Bead Size, Zirconia/Silica Beads, 3.7 g/cc Density) and centrifuged for

10 min using a tabletop microfuge. Supernatants were filtered and treated with nucleases prior to nucleic acid extraction (Sadeghi et al., 2017; Zhang et al., 2016). The viral supernatants (400 μl each) were first centrifuged at 12,000×g for 5 minutes at 4°C and supernatant then filtered through a 450nM pore size filter (Millipore, Billerica,

Massachusetts, USA) to further remove mosquito debris and bacteria. The filtrates were then treated with a nuclease mixture of 7μl of 14U turbo DNase (Ambion, Life

Technologies, Grand Island, NY, USA), 3μl of 3U Baseline-ZERO (Epicentre, Chicago,

IL, USA) and 2μl of 20U RNase One (Promega, Madison, WI, USA) in 10× DNase buffer (Ambion, Life Technologies, Grand Island, NY, USA) at 37°C for 1.5 hour to reduce background nucleic acids from host cells and bacteria. Viral nucleic acids

(protected from nuclease digestion by viral ), were then extracted from ~300 μl

resulting solutions by bead-based extraction kits (MagMAX Viral RNA Isolation Kit,

Ambion, Inc., Austin, TX, USA) without the application of DNAse to the extract (Sadeghi

et al., 2017; Zhang et al., 2016).

Viral cDNA synthesis was performed by incubation of 10 μl extracted viral nucleic

acids with 100 pmol of a primer containing a fixed 18 bp sequence plus a random

nonamer (GCCGACTAATGCGTAGTCNNNNNNNNN) at the 3′ end at 85°C for 2 min.

Next, 200U SuperScript III reverse transcriptase (Invitrogen, Waltham, Massachusetts, Page 9 of 31

USA), 0.5mM each of deoxynucloside triphosphate (dNTP), 10mM dithiothreitol, and 1× first-strand extension buffer were added to the mixture and incubated at 25°C for 10 min, followed by 50°C incubation for 1 hour. The 2nd strand DNA synthesis was performed by incubation with 50 pmol of random primer at 95°C for 2 min, 4 °C for 2min, and then with 5U Klenow Fragment (New England Biolabs, Ipswich, MA, USA) at 37°C for 1 hour. The resulting products were PCR amplified by using 5 μl of the RT-Klenow dsDNA products and 2.5 μM primer consisting of the fixed 18 bp portion of the random primer (GCCGACTAATGCGTAGTC) with 1U AmpliTaq Gold DNA polymerase (Life

Technologies, Grand Island, NY, USA), 2.5mM MgCl2, 0.2mM dNTPs, and 1× PCR

Gold buffer in a reaction volume of 50 μl. Temperature cycling was performed as follows: 1 cycle of 95°C for 5 min, 30 cycles of denaturing at 95°C for 30 sec, 55°C for

30 sec, 72°C for 1.5 min (33, 34). An additional extension for 10 min at 72°C was added to the end of the run. Using the random RT-PCR product DNA as target library preparation was performed using the Illumina XT DNA Sample Preparation Kit (Illumina,

San Diego, CA, USA) as previously described (Li et al., 2015b; Sadeghi et al., 2017;

Zhang et al., 2016) with double index barcode labeling and a 15 cycle PCR according to the manufacturer’s protocols. Library concentration was then measured using the KAPA library quantitation kit (KAPABIOSYSTEMS) The resulting libraries of single-stranded

DNA fragments were sequenced using the HiSeq 4000 Illumina platform were performed by using 2×250 cycle HiSeq Reagent Kit v2 (Illumina, San Diego, CA, USA).

The number of reads for each library is shown (Supplemental table 1b). Page 10 of 31

Sequence analysis. Sequencing datasets were processed with the goal of taxonomically assigning non-mosquito genome reads (Deng et al., 2015b). Briefly, paired-end reads of 250 bp generated by HiSeq were binned using their dual barcoding which were then removed using Illumina vendor software. Using an in-house analysis pipeline running on a 36-nodes Linux cluster, bacterial and Culex reads were subtracted by mapping to bacterial nucleotide sequences and Culex quinquefasciatus strain JHB genome (accession number NZ_AAWU00000000.1) using bowtie2 (Langmead and

Salzberg, 2012). Reads were considered duplicates if base positions 5 to 55 were identical and one random copy was kept. Low sequencing quality tails were trimmed using Phred quality score 20 as the threshold. Adaptor and primer sequences were trimmed using the default parameters of VecScreen (Ye et al., 2006). After removal of low quality and reads <50 bases long, a total of 1 to 43 million reads remained per pool which were then de-novo assembled separately for each pool using

EnsembleAssembler (Deng et al., 2015b). The contigs, together with all singlets, were then translated in all six frames and hypothetical protein sequences were aligned to a viral proteome database derived from GenBank’s virus RefSeq using BLASTx with initial

E-value cutoff of 0.01. The hits to virus-like sequences were then aligned to an in-house non-virus-non-redundant (NVNR) universal proteome database using DIAMOND

(Buchfink et al., 2015). Hits with more significant E-value to NVNR than to virus were removed. The coverage of each putative target viral genome and amino acid and nucleic acid sequences were aligned using Geneious software (version 10.00,

Biomatters, San Francisco, CA, USA). Page 11 of 31

Viral genome and phylogenetic analyses. Naming of divergent virus genomes (n=53) was based on the geographic locations from which their genomes were generated.

Other viral genomes used in phylogenetic analyses were obtained from the NCBI

GenBank database. The best-fit DNA or protein model for each alignment was selected using MEGA analysis tools. Alignments were visually inspected and misaligned regions were removed from downstream processing. Maximum Likelihood (ML) trees were generated using 1000 bootstrap replicates via the MEGA program.

Genome organization

For known viruses genomes open reading frames (ORFs) were generated based on those from the identical reference virus genomes. For newly identified virus genomes, the predication of the potential ORFs was based on those from the related reference virus genomes in the GenBank sequence database . The annotation of ORFs was first based on comparisons against the conserved domain and then against the non- redundant protein database. The remaining proteins were characterized by predicting their primary protein structure using the programs Geneious software (version 10.00,

Biomatters, San Francisco, CA, USA).

Accession numbers. The raw sequence reads generated in this study are available at the NCBI Sequence Read Archive (SRA) database under BioProject accession

PRJNA391715. All virus genome sequences generated in this study have been deposited in GenBank under the accession numbers: GenBank MH188000-MH188054. Page 12 of 31

Results

410 pools of mosquitoes collected from 23 counties throughout California were

combined into 51 larger pools for metagenomics analysis (Supp. Table 1A, B). After

homogenization, filtration, and nuclease treatment to enrich for viral particle-associated

nucleic acids, remaining RNA and DNA were extracted, reverse transcribed, randomly

PCR amplified, and converted to Illumina compatible DNA using the Nextera XT

transposon-based method. Libraries were sequenced using the Illumina HiSeq 3000

platform. Contigs were generated from each of the 51 libraries separately using de novo

assembly software Ensemble (Deng et al., 2015a). Viral sequences were identified

based on similarity of virtually translated protein sequence to all known viral protein

sequences in the virus RefSeq database in GenBank. Extension of some contigs was

then performed using Geneious (ver10.00) to detect overlapping single reads at the 5’

or 3’ of the contigs and reiterating the extension process. To exclude the possibility that

some of these detected viral genomes were endogenous viral elements, each was

compared against the Cx. tarsalis and Cx. quinquefasciatus genomes using BLASTn.

Virtual protein sequence similarity searches revealed the presence of 53 Culex-

associated viral genomes, 26 of which are newly described here (Table 1). These 53

viruses belong to different RNA and DNA virus groups whose nearest (if often distant)

genetic relative fall within or closest to viral families and orders listed by the

International Committee on Taxonomy of Viruses (ICTV). Several newly described or

still unclassified genera or families of DNA and RNA viruses were also identified. Page 13 of 31

Single-stranded RNA viruses (ssRNA). We characterized 43 putative ssRNA viruses, belonging to multiple taxonomic groups (Table 1). Their RNA-dependent RNA polymerase (RdRp) sequences clustered with members of the families

Alphatetraviridae, Bunyaviridae, , Flaviviridae, , Luteoviridae,

Mesoniviridae, Nodaviridae, Rhabdoviridae, , , , and the order .

Negative-sense RNA viruses. Six negative-sense RNA viral genomes, from the order

Bunyavirales and family Rhabdoviridae, were sequenced (Table 1). Among these, 2

Bunyaviruses (Culex Bunyavirus 1, strain Kern and Culex Bunyavirus 2 strain Fresno) and one Rhabdovirus (Culex Rhabdovirus, strain Kern) were related to previously described mosquito viruses (96%-98 RdRp aa identity) from Culex quinquefasciatus in

China (Li et al., 2015a), Culex pipiens in the US (Chandler et al., 2015; Coffey et al.,

2014; Cook et al., 2013; Cook et al., 2009), and Culex quinquefasciatus in Mexico

(Charles et al., 2016) respectively. The remaining 3 viruses: Culex Bunya-like virus strain Los Angeles, Culex Bunya-like virus strain Fresno, and Culex Rhabdo-like virus strain Los Angeles were more divergent relative to previously sequenced viral genomes.

Culex Bunya-like virus strain Los Angeles and Culex Bunya-like virus strain Fresno complete L gene (RdRp) were >98% nucleotide identical showing 69-70% aa identity to

Xinzhou mosquito virus, strain XC3-5, sequenced from Anopheles sinensis in China (Li et al., 2015a). Culex Rhabdo-like virus strain Los Angeles RdRp showed 69% aa identity to Tongilchon virus 1 strain A12.2676/ROK/2012 sequenced from Cx.

bitaeniorhynchus and Cx. pipiens mosquitoes in South Korea (Hang et al., 2016). Page 14 of 31

Phylogenetic analyses of RdRp sequences of these bunyaviruses and rhabdoviruses

RdRp is shown (Fig 1). The bunyaviruses fell into two families

( and )(Maes et al., 2018). Additionally, we assembled two shorter partial viral genomes with about 1000 bases showing 39-41% aa identity to members of the Bunyaviridae (Table 1).

Positive-sense RNA viruses. We assembled 12 positive-sense RNA viral genomes from the families Dicistroviridae, Iflaviridae, or Flaviviridae (Table 1).

Two viruses belonged to the family Dicistroviridae; Culex Dicistrovirus 1, strain

Fresno and Culex Dicistrovirus 2 strain Contra Costa showing 94% and 95% non- structural proteins aa identity to Aphid lethal paralysis virus, isolate KE-P9 from Kenya

(GenBank accession: LN907587.1) and Rhopalosiphum padi (aphid) virus (Moon et al.,

1998) respectively (Fig. 2). We also sequenced from the family of Dicistroviridae a partial viral genome about 1082 bases from Sonoma and Marin Counties, California, that shared 33% aa identity to Griffin dicistrovirus nonstructural protein from an unnamed source. Another contig from Kern County, California, of 1841 bases was 33% aa identical to hypothetical protein 1 of Hubei picorna-like virus 14 from a dragonfly

(Table 1).

We also assembled genomes of 4 viruses from the Iflaviviridae family. Culex

Iflavi-like virus 1 shared 59% aa identity to the RdRp of Armigeres iflavivirus sequenced from Armigeres mosquitoes in the Phillipines (Kobayashi et al., 2017). Culex Iflavi-like

virus 2 also with 59% aa identity to RdRp of Armigeres iflavivirus, and Culex IFlavi- virus

3 with 34% aa identity to RdRp of Wuhan insect virus 13 strain CC64511 sequenced Page 15 of 31 from unspecified insect in China (Shi et al., 2016). We also characterized Culex IFlavi- like virus 4 with 39% aa identity to Hubei picorna-like virus 35 strain QTM27260 from

Odonata (dragonflies) in China (Shi et al., 2016). Four other viral genomes highly similar to the Culex IFlavirus-like virus 4 were also assembled from four different Californian sites (Fig. 2). We named them Culex IFlavi-like virus 4, strain Sonoma, Napa, Contra

Costa, Kern, and Fresno (Table 1).

From the Flaviviridae family two viruses (Fig. 2), Culex Flavi-like virus strain

San Francisco and Culex Flavivirus strain San Francisco were sequenced. Culex Flavi-

like virus strain San Francisco showed 53% aa identity to Mercadeo virus isolate ER-

M10, a C6/36 cell line derived isolate in the insect specific flavivirus clade from an

unspecified Culex mosquito species from Panama (Carrera et al., 2015) and Culex

Flavivirus strain San Francisco with 99% aa identity to polyprotein of Culex flavivirus

isolated from Culex quinquefesciatus from the US (Kim et al., 2009). Phylogenetic

analyses of RdRp sequences of the dicistrovirus, iflavivirus, and flavivirus related RdRp

are shown (Fig 2).

We also found 4 new virus sequences from the families of Luteoviridae,

Tombusviridae, Tymoviridae, and Virgaviridae, single-stranded positive sense RNA

viruses known to infect plants (Table 1). Culex-associated Luteo-like virus strain Santa

Clara with 78% aa identity to Wenzhou sobemo-like virus 3 strain mosZJ35256 from

unnamed mosquitoes (Shi et al., 2016), Culex-associated Tombus-like virus strain

Santa Cruz with 48% aa identity to RdRp of Hubei tombus-like virus 28 from

Tetragnatha maxillosa spider (Shi et al., 2016), Culex Tymo-like virus strain Los

Angeles with 48% aa identity to RdRp of a Novel macula-like virus identified in Bombyx Page 16 of 31 mori cultured cells (Katsuma et al., 2005), and Culex Virga-like virus strain Los Angeles with 30% aa identity to RdRp of Xingshan nematode associated virus 2 (Shi et al.,

2016). Phylogenetic analyses of RdRp sequences of these Luteoviridae,

Tombusviridae, Tymoviridae, and Virgaviridae related RdRp are shown (Fig 3).

We also characterized a new genome distantly related to the family Astroviridae.

Culex Bastrovirus-like virus strain Fresno showed closest aa identity of 83% to the non- structural polyprotein of Bastrovirus-like virus strain VietNam Bat (GenBank:NC 032426) sequenced from bat feces from Vietnam in 2016 (Oude Munnink et al., 2016) (Table 1 and Fig. 4). Bastrovirus-like virus strain VietNam Bat has two ORFs (non-structural followed by structural proteins) on the same RNA genome reminiscent of while the Culex Bastrovirus-like virus strain Fresno segment sequenced showed a single open reading frame encoding a 1,761 amino acid polyprotein of the NS followed by a with 70% identity to the Bastrovirus structural protein.

Culex Tetra-like virus strain Riverside showed closest 29% translated aa identity to the capsid protein sequence of Nudaurelia capensis beta virus from

Tetraviridae family genus sequenced from a member of the Lepidoptera order (moths and butterflies) (Gordon et al., 1999)(Table 1). Also distantly related were the capsids of several member of the family from other Lepidoptera

(28% aa identity) as well as the capsid from a bisegmented member of the family

Nodaviridae (23% aa identity) reported in Lutzomyia genus (phlebotomine sand flies) reflecting the nearly equidistant nature of some protein described here to those of viruses from different families. Page 17 of 31

We also characterized 2 viruses from family Mesoniviridae, Houston virus,

strain Fresno and Houston virus, strain Solano with 99% RdRp identity to each other

and to Houston virus strain V3872 from Cx. quinquefasciatus in Texas (Vasilakis et al.,

2014) (Fig 5). A new virus from Nodaviridae family that we called Culex Noda-like virus

strain Riverside was also assembled showing 38% translated aa identity to the hypothetical protein gene of Beihai noda-like virus 30 strain BHJJX6144 sequenced from a hermit crab in China (Shi et al., 2016) (Fig 5).

Unclassified single-stranded RNA viruses. We characterized genomes of 11 viruses with RdRp amino acid identity of 24-98% to previously sequenced but still unclassified viral genomes. Fig. 6. shows the phylogenetic analysis of these 11 ssRNA viruses to the most closely related RdRp. Culex mosquito virus 1 strain Santa Cruz showed 24% aa identity to Diaphorina citri-associated C virus isolate CA segment RNA2 sequenced

from psyllid insect Diaphorina citri; Culex mosquito virus 3, strain Sonoma showed 50%

aa identity to Humaita-Tubiacanga virus isolate Rio sequenced from Aedes aegypti from

Brazil (Aguiar et al., 2016); Culex mosquito virus 4, strain Santa Clara showed 82% aa identity to Imjin River virus 1 strain A12.2496/ROK/2012 (Hang et al., 2016) sequenced from Culex bitaeniorhynchus from South Korea and the closely related (97% nucleotide identity) Culex mosquito virus 5, strain Santa Clara showed 89% aa identity to Wuhan mosquito virus 8 strain XC2-7 (Li et al., 2015a) sequenced from Culex tritaeniorhynchus

in China; Culex mosquito virus 6, strain Sonoma showed 24% aa identity to Culex luteo-

like virus strain mos191gb51453 (Shi et al., 2017) sequenced from Culex globocoxitus,

in Australia. Page 18 of 31

The closest relationship to sequenced viruses were Culex Biggie-like virus, strain Kern, with 99% aa identity to Biggievirus Mos11 (GenBank: KX924639) sequenced from Culex pipiens in USA and Culex Cordoba-like virus, strain Kings, with

98% aa identity to Cordoba virus strain EVG7-41B (Nunes et al., 2017) sequenced from

Culex nigripalpus in USA; Culex Negev #730-like virus strain Fresno, with 98% RdRp

identity to Negev virus #730 from Culex univitatus from Israel and closely related (98%

nucleotide identity) Culex Negev EO-329-like virus strain Fresno with 99% RdRp

identity to Negev virus strain EO-329 sequenced from Anopheles coustani from Israel

(Vasilakis et al., 2013); Culex Daeseongdong-like virus strain Kern with 98% identity to

Daeseongdong virus 2 strain A12.2549/ROK/2012 (Hang et al., 2016) from Culex

pipiens in South Korea; Culex Hubei-like virus strain Fresno with 99% aa identity to

Hubei mosquito virus 4 strain mosHB232766 sequenced from unnamed mosquitoes

(Shi et al., 2016) .

Double-stranded RNA viruses (dsRNA). The dsRNA viral genomes characterized in this study fell within the families Birnaviridae, , and (Fig 7). We sequenced two segments of Culex Y virus strain Kern with 97-98% aa identity to Culex

Y virus isolate P1-BS2010 segment A and segment B classified in the Bidnaviridae family from Culex pipiens complex in Germany (Marklewitz et al., 2012). We found two contigs from the family Totiviridae, -like California Culex mosquito virus strain A

Santa Cruz and Totivirus-like California Culex mosquito virus strain B Santa Cruz with

59% aa identity to Anopheles gambiae associated totivirus putative RdRp sequenced from Liberia (Fauver et al., 2016) and 24% aa identity to an hypothetical protein from Page 19 of 31

Hubei toti-like virus 10 strain 3mos6210 (Shi et al., 2017) sequenced from unnamed mosquitoes in China respectively.

From Partitiviridae family we sequenced Partitivirus-like California Culex mosquito virus strain Lake with 39% RdRp identity to that of Beihai partiti-like virus 1 strain BHTSS8738 from a woodlouse in China (Shi et al., 2016).

Single-stranded DNA viruses (ssDNA. We identified 9 ssDNA viral genomes including one each from the families Bidnaviridae, and , 3 distantly related to

Circoviridae, and 3 viruses from the family (subfamily ) (Fig.

8)

Bidnavirus-like Culex mosquito strain Los Angeles and Madera showed 37% aa identity to Vesanto virus isolate DrosEU38 from Drosophila melanogaster (GenBank:

KX648533). Bidnaviridae is a family of viruses with bi-segmented single stranded DNA genomes. Members of this family are known to infect invertebrates. The family

Bidnaviridae includes small isometric viruses that infect the silkworm Bombyx mori).

These viruses were once considered members of the family Parvoviridae (subfamily

Densovirinae) but their genomes differ in both size and coding strategy and have been re-classified into a separate family.

We found a virus from the family Anelloviridae (Rosario et al., 2017), - like Culex mosquito virus strain Fresno with 38% aa identity to a gyrovirus from a northern fulmar bird in California (Li et al., 2015c).

We also found 3 viruses belonging to the Parvoviridae subfamily Densovirinae

(arthropod hosts): Culex densovirus strain Madera-1 and Culex densovirus strain Los Page 20 of 31

Angeles) shopwing 96-97% NS protein identity to Aedes aegypti densovirus 2 (Sivaram

et al., 2009), and Culex densovirus strain Madera-2 with NS showing 81% identity to cockroach Blattella germanica densovirus-like virus (Ge et al., 2012). Three partial genomes (Culex -like virus strain Fresno, Culex circovirus-like virus strain

Butte, and Culex circovirus-like virus strain Shasta) were related to mosquito circovirus detected in Aedes vexans and (Fig 8). members of the circular Rep encoding small ssDNA genome (CRESS-DNA) virus group (Rosario et al., 2012; Simmonds et al.,

2017)

Double-stranded DNA viruses (dsDNA). We also identified two large contigs of

double-stranded DNA viruses from the family :. The first was Apis

mellifera filamentous virus-like Culex mosquito virus strain Los Angeles which has 80-

99% aa identity to Apis mellifera (Western honey bee) filamentous virus (AmFV)

(Gauthier et al., 2015) (Table 1). The AmFV genome is a double stranded DNA of

approximately 498,500 nucleotides (Gauthier et al., 2015) of which 340,623 nucleotides

could be assembled here. The second dsDNA virus, Musca domestica salivary gland

hypertrophy virus-like Culex mosquito strain Kern, showed 94% aa identity to Musca

domestica (housefly) salivary gland hypertrophy virus (Garcia-Maruniak et al., 2008).

Geographic distribution. The viral sequences in Table 1 were then used to quantify

the matching reads in each of the sampled locations. Supplemental Table S2 shows

that some viruses were widely distributed and found in multiple locations throughout the

state while others were detected at only a single sampled site. For example Culex Page 21 of 31

Bunyavirus 2 strain Fresno was detected in only 2 different pools while Culex IFlavi-like virus 1 was found in 20 pools collected throughout the state. The average number of viruses found per sampled site was 7.1 (range 0-15)(Table S1A). Page 22 of 31

Discussion

We used a viral metagenomics approach to characterize viral genomes in 12,058 adult

mosquitoes of Culex vector species and 232 mosquitoes from other 5 other species

from 124 locations in 28 counties throughout California (Table S1A, B). The near

complete genomes of 43 species of RNA viruses, 24 of which are newly described here,

and 16 DNA viruses (including 8 novel viruses) were characterized and phylogenetically

analyzed. These virus species fell both within and outside the genetic distance ranges

seen within current RNA and DNA viral families (Table 1). Providing reference genomes

will facilitate future in silico recognition and assembly of both closely and more distantly

related viral genomes in metagenomics datasets from other organisms including

insects. Using this 2016 dataset as baseline for comparison with future studies of the

mosquito virome from the same region will also allow changes in circulating viruses to

be detected. The possible role of newly introduced mosquito-associated viruses in

reducing mosquito populations, together with environmental changes (Danforth et al.,

2016), may then be experimentally tested using the appropriate viral challenges.

Of the 24 species of RNA viruses discovered here, 4 were from families of (+)

ssRNA viruses (Luteoviridae, Virgaviridae, Tombusviridae, Tymoviridae) currently only

known to infect plants. Some of these putative plant viruses were distributed widely (e.g.

Culex-associated Tombus like virus strain Santa Cruz) while others were more limited in

their distribution (Culex Virga like virus strain Los Angeles). Whether this reflects the

geographic distribution of the plant hosts of these viruses will require identification of

these presumed plant hosts. The detection of such putative plant viruses highlights the

difficulty in assigning host tropism in metagenomics studies which include the entire Page 23 of 31 organism, including digestive tract and environmentally exposed surfaces which might include dietary plant viruses in their diet or viruses of parasitic or commensal organisms residing inside or on mosquitoes such as nematodes, protozoans, or mites. The detection of a gyrovirus like sequence with 38% capsid amino acid identity to that of an avian gyrovirus may reflect feeding on a warm-blooded animal as previously reported for anelloviruses (Ng et al., 2011), particularly since most of the Culex mosquitoes analyzed here are highly ornithophilic (feed on birds).

Several of the viral sequences identified here shared high genetic identity with previously described genomes. Some were closely related to viral sequences identified in other Culex species or in other insects likely reflecting extended viral tropism for these genomes for example two dicistroviruses previously reported in different aphid species. The known geographic range of some viruses of previously reported genomes could also be expended such as the Houston virus (mesonivirus) previously reported in

Texas, South Korea, and Australia that was found throughout California.

Using a non-specific metagenomics approach no known human or animal arboviruses were detected in the sampled pools. WNV has been present in California mosquitos since 2003 and after an absence of 11 years SLEV was detected in 2015 while WEEV has not been reported since 2006(Herring et al., 2007; Pybus et al., 2012;

White et al., 2016) Whether any of the viruses detected here interfere with these arboviruses could not be determined since the mosquito pools selected here were RT-

PCR negative for these tree human pathogens. Large-scale metagenomics analysis of mosquito pools, already collected for specific testing of some human pathogens (in

California WNV, WEEV, and SLEV), can provide another level of bio-surveillance to Page 24 of 31 detect known, but not routinely tested for, arboviruses of possible concern to human and animal health. Furthermore, each year since 2008, inoculation of African Green monkey kidney VERO cells with ca. 1,000 pools of California collected mosquitoes that tested negative by qPCR for WNV, WEEV, and SLEV, have consistently failed to produce cytopathic effect characteristic of vertebrate-infecting viruses (data not shown).

This suggests that the viruses detected are unlikely to be arboviruses that infect vertebrates. Page 25 of 31

Competing interests

The authors declare that they have no competing interests.

Acknowledgements

ED and XD received funding from the Blood Systems Research Institute. MS received

funding from the Jane and Aatos Erkko Foundation #170046. CMB and LLC received

funding support from the Pacific Southwest Regional Center of Excellence for Vector-

Borne Diseases funded by the U.S. Centers for Disease Control and Prevention

(Cooperative Agreement 1U01CK000516). Page 26 of 31

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